Contributions to the functional
morphology of caudate skulls: kinetic
and akinetic forms
Nikolay Natchev1,2, Stephan Handschuh3, Simeon Lukanov4,
Nikolay Tzankov5,†, Borislav Naumov4 and Ingmar Werneburg6,7,8
1
Faculty of Natural Science, Shumen University, Shumen, Bulgaria
Department of Integrative Zoology, Vienna University, Vienna, Austria
3
VetCore Facility for Research, Veterinärmedizinische Universität Wien, Vienna, Austria
4
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia,
Bulgaria
5
Section Vertebrates, National Museum of Natural History, Bulgarian Academy of Sciences, Sofia,
Bulgaria
6
Senckenberg Center for Human Evolution and Palaeoenvironment (HEP), Eberhard-KarlsUniversität Tübingen, Tübingen, Germany
7
Fachbereich Geowissenschaften, Eberhard-Karls-Universität Tübingen, Tübingen,
Germany
8
Museum für Naturkunde, Leibniz-Institute für Evolutions- and Biodiversitätsforschung,
Humboldt Universität Berlin, Berlin, Germany
2
ABSTRACT
Submitted 24 May 2016
Accepted 1 August 2016
Published 20 September 2016
Corresponding authors
Nikolay Natchev,
[email protected]
Ingmar Werneburg,
[email protected]
Academic editor
Virginia Abdala
Additional Information and
Declarations can be found on
page 11
DOI 10.7717/peerj.2392
Copyright
2016 Natchev et al.
Distributed under
Creative Commons CC-BY 4.0
A strongly ossified and rigid skull roof, which prevents parietal kinesis, has been
reported for the adults of all amphibian clades. Our m-CT investigations revealed
that the Buresch’s newt (Triturus ivanbureschi) possess a peculiar cranial
construction. In addition to the typical amphibian pleurokinetic articulation
between skull roof and palatoquadrate associated structures, we found flexible
connections between nasals and frontals (prokinesis), vomer and parasphenoid
(palatokinesis), and between frontals and parietals (mesokinesis). This is the first
description of mesokinesis in urodelans. The construction of the skull in the
Buresch’s newts also indicates the presence of an articulation between parietals and
the exocipitals, discussed as a possible kind of metakinesis. The specific combination
of pleuro-, pro-, meso-, palato-, and metakinetic skull articulations indicate to a new
kind of kinetic systems unknown for urodelans to this date. We discuss the possible
neotenic origin of the skull kinesis and pose the hypothesis that the kinesis in T.
ivanbureschi increases the efficiency of fast jaw closure. For that, we compared the
construction of the skull in T. ivanbureschi to the akinetic skull of the Common fire
salamander Salamandra salamandra. We hypothesize that the design of the skull in
the purely terrestrial living salamander shows a similar degree of intracranial
mobility. However, this mobility is permitted by elasticity of some bones and not by
true articulation between them. We comment on the possible relation between the
skull construction and the form of prey shaking mechanism that the species apply to
immobilize their victims.
Subjects Animal Behavior, Biodiversity, Bioengineering, Taxonomy, Zoology
Keywords Feeding, Skull kinesis, mCT scanning, Urodela, Habitat shift, Prey shaking
How to cite this article Natchev et al. (2016), Contributions to the functional morphology of caudate skulls: kinetic and akinetic forms.
PeerJ 4:e2392; DOI 10.7717/peerj.2392
INTRODUCTION
The crossopterygian ancestors of the tetrapods had neurokinetic skulls. However, early
land vertebrate lineages lost this condition (Edgeworth, 1935; Laurin, 2010; Dutel et al.,
2013; Dutel et al., 2015). The extreme forms of skull kinesis found in some lizards, snakes,
and birds are judged to represent highly derived conditions (Iordansky, 1988; Evans, 2008).
The morphology of the skull in modern amphibians was studied in great detail
(see Wake & Deban, 2000; Iordanskiı̆, 2000; Heiss et al., 2016). Whereas gymnophionans
(see O’Reilly, 2000; Kleinteich et al., 2012) and anurans (see Iordansky, 1989) show only
minute internal skull movement as adults, the degree of development of skull kinetics
is rather different among urodeles (Marconi & Simonetta, 1988; Iordansky, 1988; Iordansky,
1989; Iordanskiı̆, 2000; Iordansky, 2001). Some species possess highly kinetic skulls during
their larval stages to perform particular feeding modes (Iordansky, 1988). In the course of
the ontogenetic development, however, the mobility between cranial bones substantially
decreases (Stadtmüller, 1936; Iordansky, 1988; Iordansky, 1989; Marconi & Simonetta,
1988). Iordanskiı̆ (2000) reported a high mobility in the rhinal section (prokinesis) of the
skull in the Southern crested newt (Triturus karelinii), the Smooth newt (Lissotriton
vulgaris), and the Common fire salamander (Salamandra salamandra). Information
concerning the design of the joints which allow prokinetic movements in these species
was lacking.
In the course of the present study, we investigated the cranial anatomy of a close
relative of T. karelinii, namely the Balkan-Anatolian crested newt, T. ivanbureschi.
Based on genetic data, this species was recently distinguished as a valid taxon from
T. karelinii (Wielstra, Baird & Arntzen, 2013; Wielstra et al., 2013; Wielstra et al., 2014).
Morphometric studies of the skull provided reliable information for specific
identification based on cranial shape characteristics of both species (Ivanovic et al., 2013;
Ivanovic & Arntzen, 2015).
After metamorphosis, S. salamandra is a terrestrial living species of similar body
size compared to that of T. ivanbureschi (see Stojanov, Tzankov & Naumov, 2011). It
belongs to the so called “true salamanders” (Salamandrinae), a group that is regarded as
sister taxon to all newt species (Pleurodelinae) within Salamandridae (see Zhang et al.,
2008; Pyron & Wiens, 2011). Some aspects of the feeding modes are rather different
between newts and the “true salamanders.” The newts capture prey both on land, via
tongue/jaw prehension (except for Pachytriton), as well as in water via suction feeding
(except for the “basal” clade, containing Salamandrina). The “true salamanders” are
adapted predominantly to terrestrial lifestyle and, as such, cannot perform suction
feeding, which is reflected in their cranial morphology (Deban, 2003). According to
Lukanov et al. (2016), the skeleton of the cranio-cervical complex is more fragile in
T. ivanbureschi when compared to that of S. salamandra. The authors predicted that
S. salamandra executes complex “shaking” or “killing” movements to subdue and
immobilize their prey (sensu Dauth, 1983; Dauth, 1986; Natchev et al., 2015). In the range
of the present study, we confirm that prediction and report on the specifics of the “prey
shaking” behavior in S. salamandra. The execution of vigorous “prey shaking” on land
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requires strengthening of the skull and the cranio-cervical connection (see Natchev et al.,
2015; Lukanov et al., 2016).
Using exactly the same scanning and 3D-reconstruction methods, we directly compare
the architecture of the skull in T. ivanbureschi and S. salamandra and comment on obvious
functional implications. On the base of our results, we discuss the potential impact of
cranial kinesis on the execution of the prey capture and manipulation in newts. We
provide the hypothesis that intracranial articulations in T. ivanbureschi affect the function
of the jaws during the fast-closure-phase (see Bramble & Wake, 1985) of the feeding
cycle (see Schwenk, 2000a).
MATERIALS AND METHODS
The European distribution of T. ivanbureschi includes the south-eastern parts of the
Balkan Peninsula with most of Bulgaria, the eastern parts of Greece, Macedonia and
Serbia, as well as European Turkey. In Asia, its range encompasses the coastlines of the
Aegean sea and the western and central coastlines of the Marmara sea up to 300 km
inwards, but it is absent in the inner parts of Anatolia (Wielstra et al., 2014). Populations
along the Asiatic Black sea coast, as well as the eastern coast of the Marmara sea, were very
recently raised to species level and designated the name Triturus anatolicus, based on
nuclear markers (Wielstra & Arntzen, 2016). In Bulgaria, T. ivanbureschi is ubiquitous
across the country up to 1,700 m above sea level, but is absent around the Danube river
and the lower parts of its tributaries (Stojanov, Tzankov & Naumov, 2011). It typically
inhabits stagnant ponds with thick vegetation and their surroundings, and feeds on a
variety of insect larvae, small crustaceans, earthworms, slugs, etc. T. ivanbureschi has
well-defined terrestrial and aquatic stages and leaves the water during summer, after the
breeding period. Most newts enter the water for hibernation during autumn, although
some (mostly juveniles) spend the winter on land (Stojanov, Tzankov & Naumov, 2011).
The specimens used in this study were caught in a pond near the village of Bistritsa in
Sofia district (42.595184 N, 23.367833 E).
S. salamandra has a wide range from Western and Central Europe southwards to the
south-eastern parts of the continent. In Bulgaria, it is ubiquitous in the mountainous
parts of the country (up to 2,350 m above sea level, usually between 800 and 1,600 m) and
is absent in Strandzha, the Black sea coast, and most of the Danube valley. It lives on land,
prefers humid forested areas and is usually nocturnal. It feeds on earthworms, slugs,
arthropods and their larvae (Stojanov, Tzankov & Naumov, 2011). The adult specimens
used in this study were caught near the village of Bov in Sofia district (43.016351 N,
23.367623 E).
Osteology of the skull was investigated using x-ray micro-computed tomography
(mCT). Two adult specimens from both investigated species were provided from the
collection of the National Museum of Natural History of Bulgaria (NMNH, Sofia).
The animals were fixed in 4% formaldehyde, washed, and preserved in 70% ethanol.
They were mounted in 70% ethanol and scanned for bone structures using a mCT35
(SCANCO Medical AG, Brüttisellen, Switzerland) with 70 kV source voltage and 114 mA
intensity. The reconstructed images (Figs. 1 and 2) were visualized via volume rendering
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n
+
m
os
f
# p pt
sq
*
°
eo
a
^
pf
pm
d
v
ps
eb-I
bb
f
#
cb-I
p
°
C
m
eo
pf
n
p
f
+
*
eo
^
#
pt
pq
2 mm
q
^
+
B
ch
ch
eb-I
sq
+ prokinetic joint # mesokinetic joint ^ metakinetic joint * pleurokinetic joint ° palatal joint
A
Figure 1 Craniocervical osteology of Triturus ivanbureschi based on µCT-reconstruction. (A) Lateral
view with (B) sagittal section, and (C) dorsal view. Cranial joints are indicated. Legend: a, atlas;
bb, basibranchial; ch, ceratohyal; cb-I, ceratobranchial I; d, dentary; eb-I, epibranchial I; eo, exoccipital;
f, frontal; m, maxilla; n, nasal; os, orbitosphenoid; sq, squamosum; pf, prefrontal; pm, premaxilla;
ps, parasphenoid; pq, palatoquadrate; pt, pterygoid; q, quadrate, v, vomer.
Natchev et al. (2016), PeerJ, DOI 10.7717/peerj.2392
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A
os
f
pf
p
sq eo
n
m
a
q
pm
v
p
eo
f
B
C
pt
ps
d
m
pf
n
f
p
eo
eb-I
sq
pq
q
2 mm
Figure 2 Craniocervical osteology in Salamandra salamandra based on µCT-reconstruction.
(A) Lateral view with (B) sagittal section, and (C) dorsal view. For abbreviations see Fig. 1.
Natchev et al. (2016), PeerJ, DOI 10.7717/peerj.2392
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Triturus ivanbureschi
Salamandra salamandra
A
0.000s
E
0.000s
B
0.244s
F
0.034s
C
0.809s
G
0.076s
D
1.034s
H
0.106s
Figure 3 Prey shaking behavior in both investigated species. Comparison of prey manipulation in
Triturus ivanbureschi (A–D) and Salamandra salamandra (E–H) using selected frames from video
sequence shots at 420 fps (T. ivanbureschi) and 1,000 fps (S. salamandra). Both species have prey in their
mouth and the mouth is so far closed in both species. Whereas T. ivanbureschi shows single side
movements of the body during prey shaking on land, S. salamandra shows a complex prey shaking
behavior.
using Drishti (Limaye, 2012). Nomenclature of skeletal elements follows Iordansky
(1989).
To investigate the specifics of prey manipulation in S. salamandra, we filmed two living
specimens each in lateral view when feeding on Tenebrio sp. larvae (Figs. 3E–3H). This study
was in compliance with the national laws of Bulgaria (collection-permit No. 520/23.04.2013)
and the international requirements for ethical attitude towards animals. The animals
were housed in the zoological laboratory at Vienna University. We produced three
films per specimen using the digital high-speed camera system Photron Fastcam-X
1024 PCI (Photron limited; Tokyo, Japan) at 1,000 fps. We used a highly light-sensitive
objective AF Zoom—Nikkor 24–85 mm (f/2,8-4D IF). Two “Dedocool Coolh” tungsten
light heads with 2 250 W (ELC), supplied by a “Dedocool COOLT3” transformer
control unit (Dedo Weigert Film GmbH; München, Germany). Feeding behaviour was
compared to that of T. ivanbureschi (Figs. 3A–3D; Lukanov et al., 2016).
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RESULTS
As typical for urodelans (see Marconi & Simonetta, 1988), the mCT-reconstructions
revealed a primarily flat skull roof composed of flat pairs of frontal and parietal bones
in both investigated species. In T. ivanbureschi, the joint between the squamosal, as
a palatoquadrate associated structure, and the parietal (Fig. 1) permits the typical
amphibian pleurokinetics (see Iordanskiı̆, 2000). In S. salamandra, these bones are firmly
attached (Fig. 2). In general, in the skull of S. salamandra, the major bone elements
are tightly sutured with each other suggesting a very limited (if any) potential for skull
kinesis (Fig. 2).
In T. ivanbureschi, the nasals and the frontals are not firmly fused and there is a
detectable gap between these elements (see Figs. 1B and 1C). These articulations appear to
permit prokinesis in this species. The contralateral calvarian plates overlap each other at
the median line of the skull, but do not suture firmly (Figs. 1B and 1C). In addition,
parietals and frontals are not firmly attached to each other and the sagittal section through
the skull reveals a gap between the bones (Fig. 1B), which appears to permit mesokinetic
movements. The construction of the joint between the exoccipital and the parietal is
stronger fused than between the other mentioned elements, but it is not fixed as in
S. salamandra, and a gap is visible between these bones (Fig. 1C).
In T. ivanbureschi, prey shaking on land is represented in single side movements of
the body (Figs. 3A–3D; Lukanov et al., 2016). In S. salamandra, however, the prey
shaking movements occurred in grouped clusters including up to five shakes. The prey
shake modus in S. salamandra is a complex behavior including series of ventrolateral
flexions of the whole body (Figs. 3E–3H), including the tail. This way, S. salamandra
is hitting and dragging the prey against the substrate allegedly inflicting severe
damages on the victim.
DISCUSSION
Our analyses revealed a unique skull construction in T. ivanbureschi consisting of
flexible articulations in the skull unknown to adult urodeles to this date. Compared to
T. ivanbureschi, the more “typical” caudate S. salamandra has a strongly sutured skull
permitting only little intracranial articulation. We suppose, however, that the net degree
of mobility of certain skull segments is similar in both species, because the kinetic
restrictions of tight sutures in S. salamandra will be neglectable due to the high elasticity
of the bones. This is possible because the skull roof and palatal bones of S. salamandra
are much thinner compared to T. ivanbureschi. In other words, the disadvantage of having
thicker bones in T. ivanbureschi, namely less elasticity, is circumvented by the presence
of kinetic intracranial joints.
The skull kinesis in adult salamandrids is usually reduced to pleuro- and prokinesis.
Iordansky (1982) and Iordansky (1989) defined prokinesis as elastic attachment between
the nasals and the prefrontals and the prefrontals and the frontals. This kind of skull
kinesis is typical for hinobiids and ambystomids, but is rare in salamandrids. Prokinesis
may occur only passive because urodeles lack specialized rhinal muscles (Iordansky, 1989;
Iordanskiı̆, 2000). In T. ivanbureschi, the construction of the joint between the nasals and
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the frontals indicates that the preorbital section of the skull roof can rotate against the
posterior cranial sections when certain elastic forces are applied. Such prokinetic junction
was reported earlier for T. karelinii (Iordanskiı̆, 2000).
In the skulls of T. ivanbureschi, in addition to prokinesis and pleurokinesis, we
discovered meso- and palatokinetic articulations (see Fig. 1). High speed films of
feeding T. ivanbureschi specimens revealed that the head actually shows intracranial
deformation visible in external view (N. Natchev, 2016, personal observations;
Lukanov et al., 2016).
Intracranial movements are known for some salamander larvae (see Deban & Marks,
2002). However, cranial kinesis is largely reduced in postmetamorphic salamanders
(Stadtmüller, 1936; Iordansky, 1988; Iordansky, 1989; Marconi & Simonetta, 1988).
According to Stadtmüller (1936), during the ontogeny in some urodelans, the cranial
kinesis is usually lost. The skull is highly kinetic in young larvae. Afterwards, the skull
becomes akinetic in older larvae, and kinetic skulls may secondarily reappear in
adults. However, the simplest explanation for the origin of the skull roof kinetics in
T. ivanbureschi would be that pro-, palato-, meso-, and eventually metakinesis are present
in the larval stages and the elastic connections are retained into adulthood. As such, we
propose that the skull kinesis in T. ivanbureschi might represent a neotenic feature.
However, the ontogenetic development of the skull has to be investigated in detail before
providing a final judgement.
The skull morphology of the close relative of T. ivanbureschi, T. karelinii, was studied by
Iordansky (1988) and Iordanskiı̆ (2000) using classical dissections and mechanical
manipulations. No mesokinetic joint was found by the author. Until recently, the
whole population of T. ivanbureschi was considered to belong to T. karelinii (see Wielstra,
Baird & Arntzen, 2013; Wielstra et al., 2013; Wielstra et al., 2014). It is possible that
T. karelinii simply lacks a mesokinetic articulation, but it is also possible that the specimen
dissected by Iordansky (1988) was fixed in a manner to harden the joints between the
frontals and the parietals. Further investigations on the skull osteology in newts from
different localities will provide data explaining whether mesokinesis is typical only for
T. ivanbureschi or whether it can be found in local groups of T. karelinii, or in the newly
described T. anatolicus (Wielstra & Arntzen, 2016).
The specialized head morphology of T. ivanbureschi (Fig. 1) indicates to a type of
skull kinesis, which is, in some regards, analogous to that of mesokinesis found in some
extant lizards (Frazzetta, 1962; Frazzetta, 1983; Frazzetta, 1986; Bramble & Wake, 1985;
Iordansky, 1989; Schwenk, 2000b; Payne, Holliday & Vickaryous, 2011; Mezzasalma, Maio &
Guarino, 2014; Montuelle & Williams, 2015). Despite not being able to provide irrefutable
evidences for metakinesis, we propose a possible kinetic connection between the
exoccipitals and the parietals in T. ivanbureschi. Kinetic junction between cranial bones
can be predicted on the base of the morphology of the joints connecting these bones (see
Mezzasalma, Maio & Guarino, 2014). In T. ivanbureschi we found, that there is a visible
gap in the joints between the exoccipital and the parietal bones (see Fig. 1B). The design of
this construction indicates on possible mobility in these connections.
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Table 1 Comparison of the duration of the jaw closing phase. Difference between means of duration of fast jaw closure cycle during aquatic and
terrestrial feeding among different salamandrid species.
Species
Life stage
Medium
N
Mean (ms)
SD
p
Source
Lissothriton vulgaris
Aquatic
Water
20
21
±4
0.193
Heiss, Aerts & Van Wassenbergh (2015)
5
28
±3
0.001
20
40
± 11
< 0.0001
20
34
±9
< 0.0001
Water
20
27.1
± 4.9
< 0.0001
20
32
± 5.4
< 0.0001
Air
20
32.6
± 7.8
< 0.0001
20
32.8
± 8.5
< 0.0001
Water
49
19.19
± 5.63
–
Air
60
13.34
± 4.22
Terrestrial
Aquatic
Air
Terrestrial
Ichthyosaura alpestris
Aquatic
Terrestrial
Aquatic
Terrestrial
Triturus ivanbureschi
Terrestrial
Heiss, Aerts & Van Wassenbergh (2013)
Lukanov et al. (2016)
However, even if metakinesis is present, we cannot consider an urodelan skull as
amphikinetic in the sense used for lizards. We propose that the convergent development of
meso- (and perhaps meta-) kinetic joints in lizards and in newts represents an adaptation
to different aspects of feeding performance.
In lizards, the amphikinetic mobility serves to increase the range of longitudinal
movements of the palate-maxillary complex in relation to the skull roof (Herrel et al.,
1999; Iordanskiı̆, 2000; Mezzasalma, Maio & Guarino, 2014). This permits an “active
kinesis” by which the jaws better fit to the grasped food item and the myovectors of the jaw
muscles work more effectively. The overall result is an improvement of the prey holding
and manipulation ability of the jaws (see Bramble & Wake, 1985; Iordanskiı̆, 2000;
Schwenk, 2000b; Montuelle & Williams, 2015). In T. ivanbureschi, the mechanism is rather
different (Lukanov et al., 2016) and might be based on a combination of pro-, pleuro-,
palatal-, meso-, and potentially metakinesis.
T. ivanbureschi uses hydrodynamic based mechanisms in underwater feeding (Lukanov
et al., 2016). Aquatic feeding newts benefit from pleurokinesis because it allows for
reaching a broader gape (Iordanskiı̆, 2000; Iordansky, 2001; Deban & Wake, 2000). When
feeding on smaller and/or more elusive prey, the increased velocity of the jaw closing
during the fast closing phase (see Bramble & Wake, 1985) will be of an advantage for a
predator. Our functional analysis indicates (Table 1) that the lack of rigid “skull table”
in T. ivanbureschi may benefit the jaw closing mechanism. Compared to other adult
newts studied so far (see Heiss, Aerts & Van Wassenbergh, 2013; Heiss, Aerts & Van
Wassenbergh, 2015), T. ivanbureschi needs significantly less time for jaw closure
(see Lukanov et al., 2016 and Table 1). A detailed investigation of the skull roof
morphology in the Danube crested newt (T. dobrogicus), the Alpine newt (Ichtyosaura
alpestris), and the Smooth newt (Lissotriton vulgaris) indicated that the skulls in these
newts lack the elastic connections between the frontals and the parietals and between the
parietals and the exoccipitals (see Kucera, 2013; Heiss et al., 2016). We propose that the jaw
closing mechanism in T. ivanbureschi is supported by elastic forces that permit faster
execution of the movements. During jaw opening, the epaxial musculature contracts and
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rotates the parietals in dorsal abduction (metakinesis) and the frontals move passively
(mesokinesis). During jaw opening, the flat bony elements of the longitudinal axis of
the mouth roof (Fig. 1) would be mechanically loaded by the activation of the powerful
dorsalis trunci muscles. When jaw closure starts, the potential elastic energy stored in the
bony elements of the mouth’s dorsal axis would be released and contributes for faster
jaw closure and potentially for deeper penetration of the teeth into the prey, as found in
small-sized lizards (see Montuelle & Williams, 2015).
The elastic connection between some elements of the skull may impact negatively the
stability of the whole construction. According to Lukanov et al. (2016), T. ivanbureschi
shows a rather simple prey shaking mode on land (Figs. 3A–3D). The shake cycles are
isolated and not grouped in clusters. T. ivanbureschi uses simple, strictly horizontal lateral
abduction of the body during the prey shaking. In the predominantly terrestrial living
salamandrid S. salamandra, the skull is almost akinetic (i.e., no true kinetic joints) and the
only moving element is the lower jaw. This design is consistent with the prey
manipulation behavior of the species. In contrast to the semi-aquatic T. ivanbureschi,
S. salamandra uses successive ventro-lateral flexions of the body and the neck during prey
shaking (Fig. 3). The predator hits and drags the prey against the substrate in rapid and
repetitive left-right alternations. This type of prey manipulation demands a rigid skull
frame and the kind of skull kinesis found herein would weaken the construction. The fact
that the bones of S. salamandra show a greater elasticity when compared to T. ivanbureschi
does not weaken this argument. The strongly sutured skull of S. salamandra permits
internal stability during prey shaking, but for the subsequent feeding cycle, the skull is
elastic enough to maintain strong biting forces.
Our results are in line with the previously published data concerning the skull
osteology in S. salamandra (for overview see Francis, 1934), which possesses a typical
amphibian non-flexible skull roof with fully fused sutures in adult stage. According to
Iordanskiı̆ (2000), the skull in S. salamandra is rhynchokinetic (i.e., the snout tip is
moveable against the rest of the skull). Our data indicate non-elastic connections
between the nasals, prefrontals, and frontals (see Fig. 2). The bones which build the nasal
region are flat and rather thin. It is possible that the mechanical manipulation
performed by Iordanskiı̆ (2000) had induced mechanical bending of the bones and no
(or only minimal) kinetic movement is allowed by the joints. In S. salamandra, the
squamosum is firmly attached to the exoccipital and pleurokinetic movements are rather
constrained (Fig. 2).
In conclusion, we have to note that in addition to histology the mCT scanning
technique is a powerful tool for studying cranial articulations in small-sized vertebrates.
In many salamandrids, for example, most of the bones building the skull are flat and thin.
Such bones can be easily bent and loaded during pincette manipulations (as used e.g.
by Marconi & Simonetta, 1988; Iordansky, 1988; Iordanskiı̆, 2000). The mechanical bending
and movement of defined skull segments against each other do not permit precise
identification of the kinetic joints. As such, for precise analysis, the cranial joints have
to be investigated on micromorphological level and need to be related to functional
requirements.
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ACKNOWLEDGEMENTS
We thank to Josef Weisgram and Martin Glösmann for material support. Madelaine
Böhme is thanked for discussion.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
The study was partly supported by the Project number: RD-08-66/02.02.2016 of the
Biology Department at Shumen University. We acknowledge support by Deutsche
Forschungsgemeinschaft and Open Access Publishing Fund of University of Tübingen. We
thank the “Fund for Support of the issue of publication in journals with Impact Factor
(IF) and Impact Rang (SJR)” at Konstantin Preslavsky University Shumen. The funders
had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Biology Department at Shumen University: RD-08-66/02.02.2016.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Nikolay Natchev conceived and designed the experiments, performed the experiments,
analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of
the paper.
Stephan Handschuh conceived and designed the experiments, performed the
experiments, contributed reagents/materials/analysis tools, prepared figures and/or
tables, reviewed drafts of the paper.
Simeon Lukanov performed the experiments, contributed reagents/materials/analysis
tools, prepared figures and/or tables, reviewed drafts of the paper.
Nikolay Tzankov analyzed the data, contributed reagents/materials/analysis tools,
reviewed drafts of the paper.
Borislav Naumov contributed reagents/materials/analysis tools, reviewed drafts of the
paper.
Ingmar Werneburg conceived and designed the experiments, analyzed the data, wrote
the paper, prepared figures and/or tables, reviewed drafts of the paper.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
This study was in compliance with the national laws of Bulgaria (collection-permit No.
520/23.04.2013).
Natchev et al. (2016), PeerJ, DOI 10.7717/peerj.2392
11/15
A special ethical approval commission does not exists and according to our legislation
(Biodiversity Act), permits are issued by the Ministry of Environment and Waters
(MoEW) for the scientists that are allowed to handle and collect vertebrates. The animals
were not sacrificed for the purposes of the study, but were provided as fixed specimens
from the collection of the Museum of Natural History (Sofia, Bulgaria) from Dozent
Nikolay Tzankov.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving
body and any reference numbers):
This study was in compliance with the national laws of Bulgaria (collection-permit No.
520/23.04.2013).
Data Deposition
The following information was supplied regarding data availability:
Our raw data are represented in the figures and tables. Table 1 represents data from
previously published studies.
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